Rocket immunoelectrophoresis, competitive enzyme-linked immunosorbent assay (ELISA), modified Harland-Ashworth method, and acid ferricyanide reducing substance assay were developed to determine the heating conditions of milk. The results from rocket immunoelectrophoresis showed that α-lactalbumin did not denature at 60℃ and 70℃ and was detected in the milk. heated at 100℃. β-Lactoglobulin denatured at 60℃ and was not detected in the milk heated at 90℃ and 100℃. The results from ELISA showed that the concentration of α-lactalbumin in skim milk sharply decreased at the heating temperatures between 80℃ and 100℃. The changes of β-lactoglobulin, α-lactalbumin and whey protein concentration obtained from immunoassays and modified Harland-Ashworth assay showed that most of the whey proteins denatured at 80℃. The concentration of acid ferricyanide reducing substance increased significantly after heating at temperatures above 80℃.